T-regulatory cells (Treg) are endowed with potent suppressive properties that hinder autoimmunity but also protect tumors. Colon cancer is driven by inflammation. Both clinical and experimental data suggest that Treg can protect against colon cancer by suppressing inflammation. However, we have shown that patients and mice with colon cancer harbor Treg that promote inflammation. Not knowing which Treg are protective and which are pathogenic is a major gap in knowledge and an obstacle to effective treatment of cancer. We have described previously unrecognized roles for Treg in the control of cancer-associated inflammation and provided evidence suggesting that in both mice with polyposis and patients with colon cancer Treg express the transcriptional factor ROR?t and gain pro-inflammatory properties. Based on these observations we propose that different Treg sub-phenotypes exist, they control complex innate-adaptive networks that characterize cancer-associated inflammation, and they can be targeted for cancer immunotherapy. Our long- term goal is to determine how Treg functions in colon cancer are altered to favor tumor progression and how they can be manipulated to favor better clinical outcomes. The central hypothesis is that Treg homeostasis in colon cancer is altered in favor of expansion of a pro-inflammatory population that has TH17 characteristics, suppresses T-cells, and promotes cancer. We will define this population by multiple methods outlined in our specific aims and test approaches for their manipulation. The rationale of this work is that once clarified cellular mechanisms that regulate inflammation can be therapeutically targeted to improve outcome with minimal risk of autoimmunity. To address our central hypothesis, we propose a retrospective study to correlate disease free survival with ROR?t positive or negative Treg infiltration in colon cancer tumors and relate this relationship to levels of inflammation in the tumor. Then in a prospective study, we will investigate reversibility of the pro-inflammatory phenotype and recovery of normal Treg functions after surgical removal of the tumor and during adjuvant chemotherapy. In parallel we will follow tumor specific T-cell response and seek to evaluate the combined information for predicting clinical outcome. Finally, we will obtain the molecular signature of ROR?t in Treg derived from colon cancer patients and test how inhibitors of ROR?t alter this signature and Treg properties. This will allow better definition of pro-inflammatory Treg and open future opportunities to identify signaling pathways that can be targeted. The proposed research is significant because it addresses the poorly understood role of Treg in cancer. Treg can suppress or promote inflammation. Understanding properties of pro-inflammatory Treg will open possibilities for specifically targeting the pathogenic Treg sub-population in inflammation driven diseases, including colon cancer. PUBLIC HEALTH RELEVANCE: Colon cancer is driven by inflammation. We have discovered different subsets of T- regulatory cells (Treg) that expand in colon cancer patients and can either suppress or promote inflammation. In this proposal we will define those Treg that promote inflammation to facilitate targeting these cells as a novel therapeutic strategy.